A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity

Abstract Visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques, and usually relies on genetic manipulation or the use of antibodies that underperform in tissue immunofluorescence. Starting from an endogenous ligand of gephyrin, a universal marker of the inhibitory synapse, we developed a short peptidic binder and dimerized it, significantly increasing affinity and selectivity. We further tailored fluorophores to the binder, yielding “Sylite”—a probe with outstanding signal‐to‐background ratio that outperforms antibodies in tissue staining with rapid and efficient penetration, mitigation of staining artifacts, and simplified handling. In super‐resolution microscopy Sylite precisely localizes the inhibitory synapse and enables nanoscale measurements. Sylite profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the midbrain and combined with complimentary tracing techniques reveals the synaptic connectivity.


Materials and methods
Unless otherwise noted, all resins and reagents were purchased from IRIS biotechnologies or Carl Roth and used without further purifications. All solvents used were HPLC grade. All water-sensitive reactions were performed in anhydrous solvents under positive pressure of argon. Statistical analysis was preformed using GraphPad Prism.

Peptide synthesis and fluorophore conjugation
Peptides were produced using standard solid phase peptide synthesis with Fmoc chemistry.
Shortly, 2-chlorotrityl resin (1.6 mmol/g) was swollen in dry Dichloromethane (DCM) for 30 min., then, the desired amino acid (AA) (1eq) and the Boc-Gly-OH (1eq) with 4 eq. of dry N,N-Diisopropylethylamine (DIEA) were added to the resin slurry. After overnight reaction at RT with agitation, the resin was capped with MeOH and washed with DCM and Dimethylformamide (DMF). Deprotection and conjugation cycles followed, where 20% piperidine solution in DMF was used to remove the Fmoc protecting group. After washes the peptide chain was elongated by adding AA (4 eq.) with Ethyl cyanohydroxyiminoacetate (Oxyma, 4 eq.) and N,N'-Diisopropylcarbodiimide (DIC, 4 eq.). Capping was done with DIEA (50 eq.) and acetic anhydride (50 eq.) in N-Methyl-2-pyrrolidone for 30 min. Coupling efficiency was monitored by measuring the absorption of the dibenzofulvene-piperidine adduct after deprotection. The peptides were cleaved from the resin using a cocktail of 90% TFA, 5% H2O, 5% Triisopropylsilane, for 4 hours at RT. The peptides were precipitated in ice-cold ether and then purified with HPLC and analyzed by LC-MS as described below.
The purified peptides or peptide dimers were conjugated with fluorophores either via NH 2terminus using N-Hydroxysuccinimide (NHS) coupled dyes or via cysteine -SH side chain using maleimide coupled dyes. Shortly, for NHS coupling 1 eq. of peptide was dissolved in DMF with 3 eq. of DIEA and a fluorophore-NHS was added (1 eq for standard peptides, 2eq. for peptide dimers) and agitated overnight at 4°C. Maleimide conjugation was done with similar stoichiometry and conditions, with pH 7.4 PBS as a solvent and a minimal addition of DMSO to facilitate dissolution.

Purification and characterization of peptides and fluorescent probes
The fluorescent probes were purified from the crude reaction mix by reverse phase HPLC using water acetonitrile gradient with 0.1% formic acid (FA). LC-MS validation was performed with similar gradient and LC-MS grade solvents. Semi-preparative HPLC was performed on Shimadzu Prominence equipped with a diode-array detector (DAD) system using a C18 reverse-phase column (Phenomenex Onyx Monolithic HD-C18 100×4.6 mm or Onyx Monolithic C18 100×10 mm). Purity and structural identity were verified using a DAD equipped 1260 Infinity II HPLC with a C18 reverse-phase column (Onyx Monolithic C18 50×2 mm), coupled to a mass selective detector single quadruple system (Agilent Technologies) in ESI+ mode.

Protein expression and purification
Gephyrin P2 splice variant E domain (amino acids 318-736) was expressed in E. Coli and purified as described earlier [1] . Concisely, the protein was purified using via Intein-tag (Chitin beads, New England BioLabs), and after self-cleavage the protein was obtained by sizeexclusion chromatography (SEC) column (HiLoad 16/600 Superdex 200pg, GE Healthcare) on an ÄKTA explorer system (GE Healthcare).

Modeling of the Sylite/gephyrin supracomplex
Generation of the Sylite bound to gephyrin E domain was carried out using the Rosetta FlexPepDock refinement protocol [2] using the crystal structure of gephyrin E domain bound to glycine receptor (GlyR) β subunit peptide (PDB ID: 4pd1) as a scaffold. Peptide residues where mutated using the Rosetta Fixed Backbone protocol to correspond to the binding sequence of Sylite. Following this process, the linker and dye were added to demonstrate the feasibility of the dimer formation.

Isothermal titration calorimetry (ITC)
Measurements were performed using an ITC200 (MicroCal) at 25 °C and 1,000 rotations per minute (rpm) stirring in PBS pH 7.4. Specifically, 40 μL of a 200 µM gephyrin E solution was titrated into the 200 μL sample cell containing 10 µM and 20 µM of Sylite and SyliteM, respectively. In each experiment, a volume of 2.5 μL of ligand was added at a time resulting in 15 injections and a final molar ratio between 1:2 (SyliteM) and 1:4 (Sylite). The dissociation constant (K D ) and stoichiometry (N) were obtained by data analysis using NITPIC, SEDPHAT and GUSSI [3] . Measurements were conducted three times for each probe and are given as mean values with their standard deviations.

Pulldowns with cellulose conjugated peptides
Cellulose membrane bound peptides were produced using μSPOT solid phase peptide synthesis [1] . After completion of the automated peptide synthesis, cellulose bound peptides side chains were deprotected with 90% TFA, 5% H 2 O, 5% Triisopropylsilane for 3 hrs at RT, followed by washing with 5×2 mL H 2 O. Afterwards, cellulose disks were left do dry overnight in a fume hood and stored at 4°C until use. For pulldowns, discs were first blocked in 2% (w/v) BSA in PBS for 1 hour at 25°C. Subsequently, one disc was incubated with 100 µL of mouse brain homogenate mixed with 100 µL of 10 mM TCEP in PBS for 45 min at 30°C.
After washing with 3×300 µL PBS, 50 µL loading buffer (NuPAGETM LDS-sample buffer, ThermoFisher Scientific) were added and incubation at 70°C for 2×5 min with a brief vortex in between followed. Samples were stored at -80°C until preparation for mass spectrometric proteomic analysis. As non-binding analogues of SyliteM and Sylite FSIGVSYPRRRRRRRRR, and (YSIGVSYPRpeg) 2 KC, respectively, were used. These sequences contain a binding-abolishing AA swap, described earlier [4] .

Mass spectrometric analysis of pulldowns
Alkylation of the eluate was achieved by reduction with 50 mM dithiothreitol for 10 min at 70°C and 650 rpm in a thermoshaker followed by addition of 2-iodoacetamide to a final concentration of 120 mM and incubation in the dark for 20 min. Afterwards, cold acetone was added in a 4.5:1 ratio and overnight incubation at -20°C followed. Then, the samples were centrifuged at 12,000 g for 20 min at 4°C. Pellets were washed with 4×1 mL of cold acetone with 5 min centrifugations at 12,000 g in between. Next, pellets were left to dry under ventilation for 10 min. eGFP-gephyrin P1 [5] and eGFP -pEGFP-C2 were a gift from Prof. Matthias Kneussel (ZMNH, Germany); Venus-gephyrin [6] and pHluorin-tagged GlyR β-loop transmembrane protein [7]

Culture and infection of primary neurons
Primary murine hippocampal neurons.
Primary murine hippocampal neurons were prepared from wildtype CD-1 mice (Jackson neurons were infected at day in vitro 1 to 5 (DIV1-5) with lentivirus driving the expression of full-length gephyrin tagged at its N-terminus with mEos2 [8] . Neurons were used for experiments after two to three weeks in culture (DIV15-21).

Cell fixation and immunocytochemistry
Neurons, COS-7 and HEK293 cells were fixed in 0.1 M sodium phosphate buffer pH7.4 containing 4% paraformaldehyde (EM grade, Polysciences) and 1% sucrose for 10-20 min at 37°C. After three rinses in phosphate buffered saline (PBS), the cells were permeabilized in PBS containing 0.1% Triton X-100 for 10 min at room temperature, rinsed again and blocked for 1 h in PBS with 3% bovine serum albumin (BSA). Primary and secondary antibodies were applied sequentially in blocking solution for 1 hour. The fluorescent probes were applied together with the primary antibody, unless otherwise mentioned.

Wide field fluorescence microscopy
Unless otherwise stated the coverslips with samples were inserted in an imaging chamber ( or Cy5 (Sylite). 10 images were acquired at a frame rate (exposure time) of 100 ms and at variable illumination intensity using a mercury lamp (Intensilight, Nikon) and neutral density filters to maximize the signal while avoiding saturation. All images in one channel were taken with constant settings to ensure comparability. n≥5.

2D image processing and analysis
Image processing and analysis were carried out using Fiji [9] (Fiji Is Just ImageJ) with JACoP [10] (Just Another Colocalization Plugin) plugin for colocalization analysis. Macros and scripts (Appendix 2) were written by V.K.
(Appendix 3). mEos2-gephyrin synaptic puncta were segmented, average intensity of individual punctum was determined and compared to the average intensity of the corresponding punctum in the far-red spectrum for either mAb7a with a secondary A647 antibody or the staining of Sylites.

Dual-color dSTORM super-resolution imaging
Neurons were fixed at DIV20 and immuno-labelled with primary rabbit anti-RIM1/2 antibody Coverslips were mounted in dSTORM buffer (Abbelight SMART-kit) on cavity slides (Heinz Herenz, No 1042001), sealed with twinsil (Picodent) and imaged. The measurements were taken from distinct samples with a sample size ≥ 3, for each group.
All three fluorophores (Cy5, A647, CF680) photo-switch under reducing and oxygen-free buffer conditions, making them suitable for dSTORM single molecule imaging [12] , which enables the localization of the emitters with sub-diffraction localization precision. Thanks to their close spectral proximity, Cy5 or A647 were excited and acquired simultaneously with CF680 in the same dSTORM buffer (Abbelight SMART-Kit) using a 640 nm laser (Oxxius), and their respective signals discriminated after single molecule localization using a spectral demixing strategy [13] . To implement spectral demixing dSTORM of SyliteD -(Cy5 or gephyrin-A647) and RIM1/2-CF680 we used a dual-view Abbelight SAFe360, equipped with two Hamamatsu Fusion sCMOS cameras and mounted on an Olympus Ix83 inverted microscope with a 100X 1.5NA TIRF objective. The SAFe360 uses astigmatic PSF engineering to extract the axial position and achieves quasi-isotropic 3D localization precision, and a long-pass dichroic mirror to split fluorescence from single emitters on the two cameras.
Single molecule localization, drift correction, spectral demixing, data visualization and cluster analysis [14] (DBSCAN) were performed with Abbelight NEO software, using a neighborhood radius eps = 150 nm and minPts = 50 minimum neighbors for the antibody labelling. To compensate for the lower number of detections generated by Sylite we adjusted the DBSCAN parameters to eps = 200 nm and minPts = 10. To measure the distance between presynaptic RIM and the postsynaptic gephyrin cluster, the centers of mass of the segmented clusters were determined in each fluorescence channels The Euclidean distance representing the average distance between the two-point clouds was then calculated for each cluster.

Brain section preparation and staining
Wildtype C57BL/6J mice (Jackson Laboratory) were transcardially perfused via the left ventricle with ice-cold phosphate-buffer saline 1x (PBS1x) followed by ice-cold 4% paraformaldehyde (in PBS 1x). Brains were then removed, post-fixed in 4% PFA for 2 hours, cryoprotected in 30% sucrose/PBS for 48-72 hours and cut on a cryostat (Leica CM1950) in 50µm coronal slices. The immunohistochemistry was performed in free floating sections.
Tissue sections were blocked with blocking solution (10% Donkey serum (Bio-rad) with 0.3% TritonX in PBS 1x) for 1 hour at RT, then fluorescent probes and primary antibodies were applied in blocking solution for 1 hour at RT, or 24/72 hours at 4°C. Then slices were washed 3 times with PBS and incubated with secondary antibody for 1 hour or 2 hours for the 24/72 hours staining protocol at RT. When no antibodies were applied the slices were incubated for 1h at RT with the probes. Labelled sections were then incubated with DAPI (1:5000) for 5 min at RT and washed again with PBS. Lastly, the sections were mounted onto a gelatin-coated slides using mowiol as the mounting medium. Following primary antibodies were used: gephyrin mouse mAb7a 1:1000 and mouse mAb3B11 1:1000. The fluorophore-tagged secondary antibody used was Alexa 555 donkey anti-mouse (1:1000).

Wide field and confocal imaging of brain sections
Wide-field 20x microscopy of brain sections was done with a Zeiss Axio Imager 2 equipped Bleaching was compensated with a linear gain increase of 30-40 V for an hour. The measurements were taken from distinct samples with a sample size of 4 for each group.

Animals
Experimental subjects were 3-to 6-month-old offspring of C57BL/6 mice with mutated

Injection of viruses and anatomical tracing
Isoflurane (cp-pharma, induction 4%, maintenance 1-2%) in oxygen-enriched air was used to anaesthetize mice fixed in a stereotactic frame (Kopf Instruments 1900 series). Eyes were lubricated with an ophthalmic ointment, and body temperature was maintained at 32-37 °C with a heatpad Fur was shaved and the incision site was sterilized with Cutasept solution before beginning surgical procedures. Local injections of 200 µL ropivacainhydrochlorid (Naropin; 5mg/mL, AspenGlobal) was injected subcutaneously before opening of the scalp.
After completion of surgery, intraperitoneal injections of meloxicam were administered to alleviate pain (30μl of 5 mg/ml, Metacam; Boehringer). A craniotomy was made at the injection site with a round 0.5mm drill bit (David Kopf). A volume of 200-300nl virus solution was pressure-injected intracranially using calibrated glass pipets (5μl microcapillary tube; Sigma-Aldrich) pulled in Narishige PC-100 connected to a PDES-02X (npi electronics).
Four weeks after injection, mice were sacrificed, transcardially perfused with 4% paraformaldehyde in PBS, brains were extracted and processed for histology as described above.
To evaluate the intra-connectivity of vlPAG GlyT2 neurons immunohistochemistry was performed in free floating sections as described above with the following primary antibody Subsequently, deconvolution using a computed PSF was applied (Huygens Professional package, Scientific Volume Imaging), and 3D volumetric representation, segmentation and modeling was done (Imaris, Oxford Instruments).

3D image processing
Confocal data was deconvoluted using a computed PSF (Huygens Professional package, Inhibitory synapse density in neurons was calculated by dividing total in-neuron gephyrin volume (voxels) by total neuron volume (voxels) in each tissue section of dmPAG. Gly+ synapse density: the total volume of gephyrin clusters (voxels) in proximity to synaptophysin (<1 μm distance) was divided by total neuron volume (voxels). Gly-synapse density: the total volume of gephyrin clusters (voxels), excluding the ones in proximity to synaptophysin, was divided by total neuron volume (voxels).

Probe development
Earlier reported dimeric gephyrin E domain binders [15,16] displayed an exceptional affinity in low nanomolar range but required two purification and synthesis cycles. To improve yields and facilitate the iterative selection we here use a double fmoc Lysin (U) building block to dimerize directly on resin. First, we designed and synthesized five tri-dioxaoctanoic acid dimerized peptides with different gephyrin binding sequence length and evaluated their binding to gephyrin E domain using isothermal titration calorimetry (Fig.S1A). The strongest binder had eight amino acid (AA) long binding sequence. Having determined the functionality of the Lysin-branched dimer we explored whether the linker type has an impact on the binding. To compare the different linker designs, we used the µSPOT [1] approach (a SPOT [17] and Celluspot [18] based peptide microarray synthesis method) for the comparison of eleven different dimeric linkers (Fig.S1B), all containing a core gephyrin binding sequence, and some containing previously described [4] affinity-enhancing mutations. Lastly, we tested how the length of the Gephyrin binding sequence influences the interaction, by truncating the eight amino acid sequence stepwise by one amino acid, to a minimum of three amino acids (Supplementary Table 1). The resulting 113 different dimeric binders and their monomeric counterparts were probed with gephyrin E domain in microarray format (Fig.S1C). We observed the most intense gephyrin signals for the binders having eight AAs (Fig.S1D,E), in line with our ITC findings. Notably, binders with a modified core binding motif had higher intensity than the wildtype motif. Next, we analyzed the impact of the different linkers. For the wildtype sequence we saw an increase in intensity for linkers 01, 05, 06, 07, 08, 09, 10, Table 2) while the monomeric binder and other dimeric binders had significantly lower intensity than the highest intensity binder FSIVGSLP10U (Fig.S1E). These differences could not be resolved with the mutated, higher-affinity binders, possibly due to on-array saturation of the binders with protein, leading to near-equal intensity readouts.

(Supplementary
Taking this into account we selected the dioxaoctanoic acid linker for the following dimeric probes since it contributes several H-bridges, improves solubility and allows effective and economic synthesis. Using microarray-based assays we have recently defined the sequence requirements for the binding of native gephyrin, by probing gephyrin binding peptide microarrays with mouse brain homogenate [1] (Fig.S1F). Guided by our findings on optimal dimer architecture we combined variants of this consensus binding motif and synthesized multiple different monovalent and dimeric peptides (Appendix 5) and conjugated them C-and N-terminally with sulfo-cyanine-5 (Cy5), Alexa Fluor 647 (A647) and rhodamine dyes (Supplementary Table 5). Cy5 and A647 are both suitable for dSTORM, with A647 being the brighter and more stable fluorophore [19] , while silicon rhodamine (SiR) is STED compatible and was shown to work in live cell assays [20] . In addition, hydrophobicity and overall charges was adjusted via N-terminal elongation of the core binding motif or addition of Arginines (Fig.S2A). Microscopy-based evaluation identified Sylite, a dimeric probe, and SyliteM, its monomeric counterpart as fluorescent probes with best correlation, brightness and probe overlap (Fig.S2) as well as high signal-to-background (Fig.S3). Notably, compared to the previously reported gephyrin probe, TMR2i [4] , Sylites show 10-and 150-fold improved contrast (Fig.1c, SyliteM and Sylite, respectively) and are fully compatible with advanced super-resolution techniques like direct stochastic optical reconstruction microscopy (dSTORM, Fig.2D-F). Figure S1. Optimization of probe sequence and multivalent architecture. A. Validation of dimer design strategy via ITC. Gephyrin E affinity of dimers having different-length binding sequences was determined. Minimal binder sequence length is ≥5 AAs. A ten-fold affinity increase is observed with the addition of, 7th and 8th amino acid to the binding sequence, the 8-mer binder having low-nanomolar affinity. B. µSPOT architecture of 113 monomeric and dimeric variants of gephyrin binding peptides. Peptides were based on ten binding sequences and eleven linker types (Supplementary Table 4). C. Representative example of the chemiluminescent gephyrin binding readout for the 113 monomeric and dimeric binders. Cellulose-conjugated peptides were printed on chips and incubated with 160pM of gephyrin E domain and subsequently detected with anti-gephyrin mAb3B11 and a secondary HRP conjugated antibody. Left and right are condition duplicates. D.

Supplementary Figures
Overview of the gephyrin binding intensities determined in microarray format. The intensities were internally normalized to the signal with highest intensity in the array and subsequently averaged and plotted for each peptide binder. E. The eight amino acid FSIVGSLP wild-type binding motif yields a higher luminescence signal than the shortened dimeric and monomeric analogues. Monomer and dimers (01-11U) based on the enhanced binder sequence YSIVGRYP have the highest intensity readout in the microarray. 08U and 09U dimers have lower readouts than the most intense binder YSIVGRYP11U. One-way ANNOVA followed up by Tukey test for multiple comparisons was performed. Mean±SEM, P<0.05. n=6. F. Native gephyrin consensus peptide-binding motif determined by screening and alignment of fine mapped gephyrin-binding sites [1] .

Figure S5. Gephyrin mAb7a explicitly binds a phosphorylated epitope. A. Representative examples (top)
and averaged intensities as density blots (bottom) of mAb7a binding overlapping gephyrin fragments. The gephyrin (GPHN-1 isoform) [21] sequence was displayed in microarray format in the form of 15 AA peptides overlapping 12 AA with and without phosphorylations (Supplementary Table 5). mAb7a antibody binding was visualized with a secondary anti-mouse HRP conjugated antibody. Top panel: boxed -triplicates of phosphorylated peptide sequences. Bottom: a positional intensity readout, boxed is the region with phosphorylated sequence replicates. Intensities normalized to the highest intensity detected in the array. B. Averaged normalized intensity readout of the boxed region in A. X represents the phosphoserine. Chemiluminescent readout reports SLSTTPSEpSPRAQAT as the primary mAb7a epitope. Thus, indicating that phosphorylation of Serin 270 is necessary and sufficient for binding while phosphorylation of Ser 268 does not appear to affect binding.  A. DIV21 cortical neurons were fixed and co-stained with Sylite (500 nM) and mAb7a. Similar pattern of antibody labeling and Sylite is observable. Scale bar 10 μm. B. Sylite staining correlates with the staining of mAb7a, but the degree of correlation is lower than with mAb3B11. These data are in-line with the narrow specificity of mAb7a, that labels only Ser270 phosphorylated neuronal gephyrin, therefore a lower correlation of mAb7a with Sylites is observed. On the "-Probe" group only a secondary mouse A488 antibody was applied. Mean ± SEM. Scale bar 10 μm. Significance determined with Mann-Whitney test.  1 -full overlap, 0 -no overlap. After 24-hour staining of brain tissue about 40% of clusters detected by the antibodies are also Sylite-positive. The number of clusters detected by Sylites is higher than the number of clusters detected with mAb3B11, hence only ~10% of Sylite-positive clusters are co-labeled with mAb3B11. After 72-hour staining the maximum degree of overlap drops to ~15%, in line with the apparent increased unspecific staining of the antibodies. Mean ± SD. C. Sylites enable ultra-rapid synapse staining and visualization. Hippocampal section co-staining with Sylite and mAb7a. Green -Sylite, gold -mAb7a, blue -DAPI nuclear staining. Left -24-hour staining. mAb7a and Sylite clusters partially overlap. mAb7a appears to have some unspecific connective tissue staining, Sylite synapse visualizations appear more consistent, a directional pattern of inhibitory synapse distribution can be observed. Right -72-hour staining. Higher background staining with mAb7a is observed, the quality of Sylite labeling does not change. Scale bar 15 μm.